Human Primary Articular Chondrocytes, Chondroblasts-Like Cells, and Dedifferentiated Chondrocytes: Differences in Gene, Microrna, and Protein Expression and Phenotype
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TISSUE ENGINEERING: Part C Volume 17, Number 2, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2010.0200 Human Primary Articular Chondrocytes, Chondroblasts-Like Cells, and Dedifferentiated Chondrocytes: Differences in Gene, MicroRNA, and Protein Expression and Phenotype Tommy A. Karlsen, M.Sc., Aboulghassem Shahdadfar, Ph.D., and Jan E. Brinchmann, M.D., Ph.D. In this study we have isolated human primary uncultured articular chondrocytes. When these cells are allowed to proliferate within their own extracellular matrix (ECM), they begin to produce hyaline ECM molecules similar to embryological chondroblasts. These cells are called chondroblast-like cells. Upon continued culture these cells spread onto the plastic surface and dedifferentiate. We have characterized these three stages of chondral cells by gene expression and expression of microRNAs (miRNAs) and proteins. Gene expression was quantified by real- time reverse transcriptase (RT) polymerase chain reaction, miRNA expression by miRNA arrays, and protein synthesis by extra- and intracellular flow cytometry. Many of the genes, miRNAs, and proteins were differentially expressed in the different stages of chondral cells. In the context of cellular therapy, expression of some genes is a cause for concern. The best source of cells for treatment of lesions of hyaline cartilage has not yet been identified. Adult chondroblast-like cells may be strong candidates. Profound understanding of how expression of genes and synthesis of proteins are regulated in these cells, for instance, by miRNAs, may reveal new strategies for improving their synthesis of hyaline ECM. This insight is important to be able to use these cells in the clinic. Introduction the features of embryological chondroblasts, they are called chondroblast-like cells (CBLC). utologous chondrocyte implantation (ACI) is now Although the use of CBLC in clinical transplantation Astandard therapy in many centers for patients with focal might well improve on the clinical results obtained by using lesions of hyaline articular cartilage.1,2 ACI requires the re- dedifferentiated chondrocytes, this has not yet been dem- moval of a small piece of articular cartilage, followed by onstrated clinically. We are presently conducting clinical ex vivo expansion of its content of articular chondrocytes trials using these cells. However, to further improve cell (AC) to obtain large number of cells for implantation. Ex vivo therapy strategies it is important to fully understand the expansion of AC in monolayer cultures invariably leads to biology of the cell populations used. This includes quantifi- dedifferentiation of the chondrocytes.3,4 This means that the cation of the genes expressed, the proteins they encode, and AC revert to a less specialized cell type, where synthesis of the understanding the mechanisms that regulate gene expres- hyaline cartilage-associated proteins type II collagen (COL2) sion. MicroRNAs (miRNAs) are a recently discovered class and aggrecan (ACAN) is replaced by synthesis of COL1 and of molecules that affect both mRNA expression and protein versican (VCAN), respectively.5 These extracellular matrix synthesis. miRNAs are small single-stranded RNA molecules (ECM) components are typical of fibrocartilage.6,7 Trans- (*21–22 nt). miRNAs are derived from long primary tran- plantation of dedifferentiated chondrocytes frequently leads scripts (pri-miRNA, 2 kb in length) that are mainly tran- to the formation of fibrocartilage, or a mixture of fibrocartilage scribed by the RNA polymerase II. In the nucleus the and hyaline cartilage within the lesion.8,9 It is believed that this pri-miRNAs are processed by the RNase Drosha to create repair tissue has inferior functionality and durability, and that short stem-loop molecules called pre-miRNAs (*70 nt) that an end result may be arthrosis in that joint.10 Most likely, a cell are exported to the cytoplasm. In the cytoplasm the pre- population more similar to the AC found in vivo is needed for miRNAs are further processed by the cytoplasmic RNase implantation to improve the outcome of ACI. Recently, we Dicer into a 21–22 nt miRNA duplex. The miRNA duplex is established a new culture system where AC were cultured in then assembled into the RNA-induced silencing complex their own ECM.11 At day 7–10 of culture the cells maintained a where one of the strands (mature miRNA) base-pair with rounded shape and expressed genes for several hyaline ECM complementary mRNA transcripts.12,13 It is thought that molecules while still proliferating. As these cells have many of perfect complementary leads to degradation of the mRNA, Institute of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway. 219 220 KARLSEN ET AL. whereas imperfect complementary results in translational systems, Abingdon, United Kingdom). Relative quantification inhibition.14,15 It is generally accepted that the main function was performed using the 7300 Real-Time reverse transcriptase of miRNA is to knock down gene activity, although it has (RT) PCR system (Applied Biosystems) and TaqManÒ Gene been shown to activate transcription also.16 One-third of all Expression assays following protocols from the manufacturer human genes have been estimated to be regulated by miR- (Applied Biosystems). TaqMan assay numbers are listed in NA.17 As expression of individual miRNA relatively easily Table 1. All samples were run in technical triplicates contain- may be either increased or decreased by introducing miRNA ing 1.0 mL cDNA in a total volume of 25 mL. The thermocycling precursor molecules or antagonists,18 insight into expression parameters were 958C for 10 min followed by 40 cycles of 958C of miRNA in the different types of chondral cells may in- for 15 s and 608C for 1 min. Validation of 32 different endog- troduce a new strategy for enhancing expression of genes enous controls was performed using the TaqMan Human expressing ECM molecules or decreasing expression of genes Endogenous Control Plate following protocols from the and proteins associated with dedifferentiation. manufacturer (Applied Biosystems). Subsequently, all sam- The objective of this study was to investigate the three cell ples were scaled relative to expression level of CDKN1A, populations: primary uncultured chondrocytes, CBLC, and which was found to be one of the most suitable endogenous dedifferentiated chondrocytes with respect to their expres- controls in our experimental settings. sion of a large number of genes, proteins, and miRNA related to chondrogenesis. The results add to our under- miRNA array and real-time RT polymerase chain standing of the biology of in vitro chondrogenesis, and may reaction validation also introduce new strategies for manipulation of chon- Total RNA enriched with small RNA molecules (including drocytes used in clinical protocols. miRNA) was extracted from the cells using the Trizol (In- vitrogen, Carlsbad, CA) procedure with the following modifi- Material and Methods cations: 1 mL of isopropanol was included in the precipitation Chemicals step and the samples were kept at À808C for minimum 30 min. After centrifugation the RNA pellet was dissolved in Trizol and All chemicals were purchased from Sigma-Aldrich (St. column-purified using reagents and following protocols from Louis, MO) unless otherwise stated. the miRNeasy kit (Qiagen, Hilden, Germany). After DNase treatment (Ambion), the total RNA was Isolation and culture of the cells quantified by spectrophotometry (Nanodrop). miRNA pro- Articular cartilage was obtained from the femoral surface of filing was obtained through the miRNA microarray service, three knees obtained from legs amputated for reasons other including in depth analysis, from LC Sciences (Houston, TX, than cartilage disease. The donors were 84, 79, and 76 years of www.lcsciences.com). Analysis of variance ( p < 0.01) was age, and all provided written informed consent. The study was used for clustering to make a heat map. The arrays were approved by the Regional Committee for Medical Research based on the Sanger miRBase Release 13.0 and contained 875 Ethics, Southern Norway, Section A. The cells was isolated as unique probes. For real-time reverse transcriptase (RT) previously described.11 Briefly, all the articular cartilage with polymerase chain reaction (PCR) validation, the miRNAs normal and healthy appearance was harvested and cut into were reverse transcribed to cDNA using primers included in small pieces. Only for one donor (donor 2) there were minor the TaqMan miRNA assays (Table 1) and reagents from the areas of the cartilage with abnormal appearance. This patient TaqMan miRNA RT kit following protocols from the man- had no previous history of osteoarthritis in the knee. The car- ufacturer (Applied Biosystems). Relative quantification was tilage pieces were digested with Collagenase type XI (1.2 mg/ performed using the same parameters as described above for mL) at 378C for 90–120 min. The ensuing structures, called the real-time RT PCR analysis. CDKN1A was used as en- chondrocytes in autologous ECM, were washed several times dogenous control. The prediction tools PicTar (http://pictar. and resuspended in culture medium consisting of Dulbecco’s mdc-berlin.de/), TargetScan (www.targetscan.org/), miRDB modified Eagle’s medium/F12 supplemented with 10% fetal (http://mirdb.org/), Diana Micro-T (http://diana.cslab.ece. bovine serum, 50 mg/mL ascorbic acid, 100 units/mL penicil- ntua.gr/), and miRNA viewer (http://cbio.mskcc.org/ lin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B. mirnaviewer/)